Heterogeneity of porcine bone marrow-derived dendritic cells induced by GM-CSF
Authors:
Sang Eun Kim aff001; Jeong Ho Hwang aff001; Young Kyu Kim aff001; Hoon Taek Lee aff003
Authors place of work:
Department of Animal Biotechnology, Konkuk University, Gwangjin-gu, Seoul, Republic of Korea
aff001; Animal Model Research Group, Jeonbuk Department of Inhalation Research, Korea Institute of Toxicology, Jeongeup, Jeollabuk-do, Republic of Korea
aff002; Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Gwangjin-gu, Seoul, Republic of Korea
aff003
Published in the journal:
PLoS ONE 14(11)
Category:
Research Article
doi:
https://doi.org/10.1371/journal.pone.0223590
Summary
In vitro generation of dendritic cells (DCs) is advantageous for overcoming the low frequency of primary DCs and the difficulty of applying isolation techniques for studying DC immunobiology. The culture of bone marrow cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used extensively to generate bone marrow-derived dendritic cells (BMDCs). Studies have reported the heterogeneity of cells grown in murine GM-CSF culture based on the levels of MHCII expression. Although porcine DCs are generated by this classical method, the exact characteristics of the BMDC population have not yet been defined. In this study, we discriminated GM-CSF-grown BMDCs from gnotobiotic miniature pigs according to several criteria including morphology, phenotype, gene expression pattern and function. We showed that porcine BMDCs were heterogeneous cells that differentially expressed MHCII. MHCIIhigh cells displayed more representative of DC-like morphology and phenotype, including costimulatory molecules, as well as they showed a superior T cell priming capacity as compared to MHCIIlow cell. Our data showed that the difference in MHCIIhigh and MHCIIlow cell populations involved distinct maturation states rather than the presence of different cell types. Overall, characterization of porcine BMDC cultures provides important information about this widely used cellular model.
Keywords:
Gene expression – Phenotypes – T cells – Flow cytometry – Phagocytosis – Swine – Bone marrow cells – Phagocytes
Introduction
Dendritic cells (DCs) are components of the immune system that can present antigens to T cells [1]. Conventional DCs (cDCs) provide signals for T cell activation and differentiation, and are therefore regarded as professional antigen-presenting cells (APCs) of the immune system [2]. However, study of these essential cells has been complicated by the low frequency of DC populations in blood and tissue. For this reason, the biology of DCs has been studied in cells grown in vitro from hematopoietic precursors, in the presence of growth factors [3]. Besides, in vitro generated DCs have been designated as cell-based vaccines for immunotherapy [4]. Bone marrow cells (BMCs) have been cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine involved in the development and homeostasis of mononuclear phagocytes, to generate bone marrow-derived dendritic cells (BMDCs) that resemble tissue DC [5, 6].
In bone marrow cultures induced by GM-CSF, CD11c+ MHCII+ cells have been assumed to be the source of pure BMDCs, whereas macrophages are thought to be adherent cells [3, 7]. However, the studies reported that this classical method produces heterogeneous populations of murine myeloid cells in non-adherent populations and loosely adherent populations [8–10]. The study suggested that MHCIIhigh cells, which were previously shown to be DCs and MHCIIlow cells, closely resemble macrophages in the murine GM-CSF-derived heterogeneous population. Other studies suggested that MHCIIlow cells contain immature DCs, which further upregulate MHCII on their surface, indicating maturation in mice [11, 12].
The porcine immune system is similar to the human immune system with respect to DC biology [13, 14]. The gnotobiotic miniature pig is the best model to study immunology, including immune cell ontogeny, microbial infection, and xenotransplantation [15–18]. To study porcine DC biology, in vitro differentiated DCs have been widely used [19], especially BMCs are cultured with GM-CSF for generation of BMDCs likewise other species [20]. The non-adherent cells have been considered as pure BMDCs and are characterized by expression of the surface molecules, CD1, CD16, CD80/86, CD172a, and MHC class II [21]. However, it is unclear whether porcine BMDCs are heterogeneous like murine BMDCs.
In this study, BMCs were isolated from gnotobiotic miniature pigs and cultured with GM-CSF to generate DCs. We classified GM-CSF-grown porcine BMDCs into MHCIIhigh cells and MHCIIlow cells, in a similar manner as murine BMDCs. These two populations from non-adherent cells were characterized according to their morphology, phenotype, gene expression profile, and function. On the basis of these characteristics, we showed that non-adherent cells isolated from GM-CSF-grown BMC cultures were heterogeneous in terms of their levels of MHCII expression. Therefore, these findings of GM-CSF-derived porcine BMDCs could lead to improvements in our understanding of the porcine immune system.
Materials and methods
Animals
Gnotobiotic miniature pigs were kept under absolute barrier contained facility at the Bio-organ Research Center of Konkuk University, Seoul, Republic of Korea [22]. Animal experiments were carried out based on the National Institutes of Health guidelines for the care and use of laboratory animals. The study was conducted after obtaining approval from the Institutional Animal Care and Use Committee (IACUC) of Konkuk University (KU16168). In this study, three, 3-week-old piglets were used: K8082-1, K8082-2, and K8083-4. The animals were sacrificed using CO2 according to IACUC guidelines, and then the humerus, tibia, and femur were collected to isolate BMCs.
Cell preparation
The BMDCs were generated using a previously described method with some modifications [20]. The BMCs were cultured for 10 days at a density of 5 × 105 cells/mL in RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM minimal essential medium, non-essential amino acids (Gibco), and 100 ng/mL of porcine GM-CSF (R&D Systems, Madrid, Spain). GM-CSF was additionally supplemented on days 2, 4, and 6. Differentiated cells were obtained from the non-adherent cell population after day 10. The cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air.
Flow cytometry analysis
The cells were incubated with monoclonal antibody from hybridoma culture supernatants for 30 min, and then washed twice with Dulbecco’s phosphate-buffered saline (DPBS; Welgene, Seoul, Republic of Korea). The cells were incubated with secondary antibody and washed twice with DPBS. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Biolegend, San Diego, CA, USA) and allophycocyanin (APC)-conjugated goat anti-mouse IgG (Biolegend) were used as secondary antibodies. The cells were resuspended in 500 μL fluorescence-activated cell sorter buffer (5% FBS in DPBS) and flow cytometry analysis was performed on a BD Accuri™ C6 flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The following antibodies were used: anti-porcine CD1 (HB140), anti-porcine CD172a (HB142), anti-porcine CD16 (G7), anti-porcine CD11b/CD18 (PM3-15), and anti-porcine MHCII (MSA3) from hybridoma culture supernatants. Anti-porcine CD14 ascites, APC-conjugated anti-human CD86 (IT2.2; Biolegend), APC-conjugated anti-human CD163 (GHI/61; Biolegend), and anti-porcine CD117 (2B8/BM; Bio-Rad, Hercules, CA, USA) were also used.
To isolate MHCIIhigh and MHCIIlow populations from BMDCs and c-kit+ hematopoietic stem cells (HSCs) from BMCs, the cells were sorted by a FACSAria™ instrument (Becton Dickinson). Flow cytometry analysis was conducted using FlowJo software (https://www.flowjo.com/).
RNA sequencing
Total RNA was extracted from sorted cell subsets including c-kit+ HSC, MHCIIhigh, and MHCIIlow cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). In order to construct cDNA libraries with the TruSeq RNA library kit (illumine, San Diego, CA, USA), 1ug of total RNA was used. The protocol consisted of polyA-selected RNA extraction, RNA fragmentation, random hexamer primed reverse transcription and 100nt paired-end sequencing by Illumina HiSeq2500 (illumine, San Diego, CA, USA). The libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide and qualified using an Agilent Technologies 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). We processed reads from the sequencer and aligned them to the Sus scrofa using Tophat v2.0.13 [23]. Transcript assembly and abundance estimation using Cufflinks v2.2.1 [24].
The transcript-level relative transcript abundances were measured in FPKM (Fragments Per Kilobase of exon per Million fragments mapped) using Cufflinks. We performed the statistical analysis to find differentially expressed genes (DEG). Filtered data were log2-transformed and subjected to quantile normalization. For DEG set, Hierarchical clustering analysis was performed using complete linkage and Euclidean distance as a measure of similarity. Gene-enrichment and functional annotation analysis for significant gene list was performed using Gene Ontology (http://geneontology.org/). We used multidimensional scaling (MDS) method to visualize the similarities among samples. We applied to the Euclidean distance as the measure of the dissimilarity.
Real-time polymerase chain reaction
The cDNA was reverse-transcribed from total RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Synthesized cDNA was denatured at 95°C for 10 min and amplified using SYBR Premix Ex Taq II (Takara, Kusatsu, Japan) on an Applied Biosystems 7500 Real-Time PCR System cycler, with 40 cycles of 95°C for 15 s and 60°C for 1 min. All data were acquired as ΔCt values and automatically converted to double delta Ct (ΔΔCt) values by 7500 software (Applied Biosystems). The value of 2−ΔΔCt was calculated to obtain expression fold-change data. Primers specific for CD86, CD40, IFR4, CCR7, FcεR1α, CSF1R, CD163, and CD117 (S1 Table) were used.
Mixed lymphocyte reaction
For the preparation of allogeneic T cells, splenocytes were isolated and incubated for 2 h to remove attached cells. Floating splenocytes were harvested and labelled with carboxyfluorescein succinimidyl ester (CFSE; Invitrogen). CFSE-labelled allogenic splenocytes were co-cultured with APCs (MHCIIhigh and MHCIIlow cells) for 5 days. Then, 105 splenocytes were mixed with APCs according to the desired APC:splenocyte ratio (1:2, 1:6, 1:18, 1:54 and 1:162) in 96-well U-bottom plates. For gating proliferating population, CFSE-stained splenocytes was checked their proliferation at 24h and confirmed without proliferation (data not shown). T cell proliferation was examined using flow cytometry and analyzed by FlowJo software.
Phagocytosis assay
The cells were seeded at 2 × 105 cells and incubated with latex beads coated with FITC-labelled rabbit IgG (Cayman Chemical, Ann Arbor, MI, USA) for 30 min, 1 h, 2 h, 3 h, and 4 h in 96-well U-bottom plates. To distinguish cells that were phagocytosed from those simply binding to the beads at the surface, a short (1–2 min) incubation with Trypan Blue dye quenching solution, followed by a wash with assay buffer, was used to quench the surface FITC fluorescence. Phagocytosed cells were detected using flow cytometry and analyzed by FlowJo software.
Results
Heterogeneity of the GM-CSF-derived BMDCs
To generate BMDCs in vitro, BMCs were obtained from 3-week-old gnotobiotic miniature pigs and cultured with GM-CSF supplementation. To enrich BMDCs, we harvested non-adherent cells from the GM-CSF culture and confirmed the MHCII expression of these cells. The non-adherent cells were comprised of two distinct populations (MHCIIhigh and MHCIIlow) based on the MHCII expression (Fig 1A). Adherent cells were mainly composed of MHCIIlow population (S1 Fig). We employed FACS sorting to purify MHCIIhigh and MHCIIlow populations that could not be distinguished by adhesion properties. In the two populations, differences in morphology were observed; the MHCIIhigh cells had a more dendritic morphology, and showed cluster formation, relative to the MHCIIlow cells (Fig 1B). Thus, MHCIIhigh cells were more representative of DC-like morphology than MHCIIlow cells. From these results, two populations were isolated from non-adherent cells from porcine BMDC cultures, based on different expression levels of MHCII, in a similar manner to murine cells.
Surface marker expression levels of MHCIIhigh and MHCIIlow cells
Because there were differences in MHCII expression and morphology, MHCIIhigh and MHCIIlow cells were sorted to confirm their different phenotypes (Fig 2). We examined CD86, CD1, CD16, CD11b/CD18, CD172a, CD14, and CD163 to clearly define distinct populations. The MHCIIhigh and MHCIIlow cells expressed CD172a and CD14, indicating that they both differentiated into myeloid lineages. We observed high expression of the porcine DC markers, CD86, CD1, and CD16, in MHCIIhigh cells, and low expression in MHCIIlow cells. Complement receptor CD11b/CD18 and scavenger receptor CD163, which are highly expressed on activated myeloid cells, were found to be highly expressed on MHCIIhigh cells, but they showed very low expression on MHCIIlow cells. The immature DC phenotype involved intermediate or low expression of MHCII and costimulatory molecules such as CD86, together with high expression of CD14. These results suggested that MHCIIlow cells resembled immature DCs, and MHCIIhigh cells underwent spontaneous maturation and expressed higher amounts of the same markers.
Gene expression patterns of MHCIIhigh and MHCIIlow cells
For gene expression analysis, we sorted c-kit+ HSCs from BMCs and the two populations (MHCIIhigh and MHCIIlow cells) described in Fig 1A, followed by mRNA sequencing. Using gene hierarchical cluster mapping and MDS analysis, each cell type from the three different piglets clustered together, confirming that the transcription profiles of each cell type were similar (Fig 3A and 3B). We then confirmed that 368 genes were differentially expressed between MHCIIhigh and MHCIIlow cells. However, the MHCIIhigh and MHCIIlow cell populations were close together, suggest that these they are not the completely separated as distinct cell type unlike what has been described for mice and humans [8, 9]. We therefore hypothesized that MHCIIhigh and MHCIIlow cell populations were comprised of BMDCs in distinct maturation states, as opposed to different cell types being present.
To further explore this possibility, DC-related gene expression patterns were investigated in each cell type. Transcriptome analysis revealed that MHCII (SLA-DR) and costimulatory molecules (CD40, CD80, CD83, and CD86) were highly expressed in MHCIIhigh cells that were induced during BMDC differentiation (Fig 3C). In addition, receptors (CD163, MRC1, FCER1A, FCGR2B, and TLRs) involved in the DC innate immune response were also highly expressed in MHCIIhigh cells. MHCIIlow cells expressed more CD34, CD59, CD177, Sox4, and Foxo1, which showed the highest expression levels in HSCs. These data showed that MHCIIhigh cells highly expressed genes related to the DC signature and innate immune response, whereas, genes enriched in HSCs were found to be expressed more in MHCIIlow cells than MHCIIhigh cells.
To determine the difference between MHCIIhigh and MHCIIlow cells, DC-related gene expression patterns were validated by qPCR. As expected, the CD86 and CD40 costimulatory molecules were expressed at high levels by MHCIIhigh cells, and to a lesser extent by MHCIIlow cells (Fig 3D). In addition, the IRF4 transcription factor, which controls the development of BMDCs falling within the mature gate, was highly expressed on MHCIIhigh cells compared to MHCIIlow cells. MHCIIhigh cells also expressed higher amounts of CCR7, FcεR1α, CD163, and CSF1R, whereas MHCIIlow cells showed higher expression of CD117. Consistent with these results, activated DC-related genes were highly expressed on MHCIIhigh cells compared to MHCIIlow cells. Therefore, we assumed that the two populations (MHCIIhigh and MHCIIlow cells) had cell-to-cell variations that were the result of different states of BMDC maturation.
Functions of MHCIIhigh and MHCIIlow cells
To examine the ability of DCs that can stimulate T cells as professional APCs, the mixed lymphocyte reaction was conducted using allogeneic splenocytes co-cultured with sorted MHCIIhigh and MHCIIlow cells (Fig 4). When sufficient APCs were supplied to expand splenocytes (the APC:splenocyte ratio was 1:2 ~ 1:18), the MHCIIhigh and MHCIIlow cells displayed comparable ability to stimulate T cells. There was no significant difference in the T cell proliferation ability of MHCIIhigh and MHCIIlow cells. In contrast, when given a lower number of APCs to stimulate T cells (APC:splenocyte ratio, 1:54 ~ 1:162), MHCIIlow cells were inferior in terms of proliferating T cells compared to MHCIIhigh cells. Accordingly, MHCIIhigh cells showed superior T cell priming capacity compared to MHCIIlow cells.
DCs are mononuclear phagocytes; therefore, to analyze phagocytic abilities, MHCIIhigh cells and MHCIIlow cells were cultured with phagocytic beads (Fig 5). MHCIIhigh cells had more uptake of phagocytic beads during 3 h (Fig 5A; 50% of MHCIIhigh cells and 37% of MHCIIlow cells). When cells were incubated with phagocytic beads, MHCIIlow cells were significantly less efficient at phagocytosis, as expected (Fig 5B). Together, these results indicated that MHCIIhigh cells are more functionally activated DCs than MHCIIlow cells, because they had superior T cell-priming ability and phagocytic ability.
Discussion
The in vitro generation of DCs in culture is advantageous for studying DC biology. In particular, GM-CSF, a hematopoietic growth factor, has been used to supplement BM cultures to generate CD11c+ MHCII+ cells, which are often termed BMDCs [5]. From the GM-CSF BM culture, DCs have been enriched from non-adherent cells, whereas adherent cells are thought to be macrophages. In addition, the studies of the discrimination of murine BM cultures showed the heterogeneity of GM-CSF-derived non-adherent cells and loosely adherent cells [8, 9]. They suggested that the MHCIIhigh cell population, considered as a DC and MHCIIlow cell population, actually corresponded to macrophages from murine BM cultures. However, another study showed that GM-CSF culture induced differentiation towards immature and mature cDC2s, which were shown to be efficient at promoting Th17, as well as Th2 immune responses, in a non-adherent population [11].
The pig has been considered an important large animal model, and gnotobiotic miniature pigs are probably the best model for studying immunology [18]. The porcine immune system resembles the human immune system with respect to DC biology, because their gene expression signature for cDC2 is close to the human counterpart [14, 25, 26]. The classical protocols for generating in vitro DCs in humans and mice are similar to the porcine method. Although GM-CSF-generated porcine BMDCs have been widely used, the heterogeneity of the cells has not been defined. Considering that murine GM-CSF cultures often provide two populations based on MHCII expression level, we discriminated GM-CSF-grown BMDCs from gnotobiotic miniature pigs based on several criteria.
In this study, we noted heterogeneity in the non-adherent cells from gnotobiotic miniature pigs according to their MHCII expression levels (MHCIIhigh and MHCIIlow cells). It has been reported that cells developing in porcine GM-CSF culture were also heterogeneous, as in murine cultures. Although both populations showed DC-like morphology, MHCIIhigh cells had a more dendritic morphology, and showed cluster formation, relative to MHCIIlow cells. The phenotype analysis showed that MHCIIhigh cells displayed a DC-like phenotype that involved CD86+, CD1+, CD16+, CD11b/CD18+, CD172a+, CD14low, and CD163+. MHCIIlow cells also expressed these DC markers; however, they had low expression levels of CD86, CD1, CD16, CD11b/CD18, and CD163, and higher CD14 expression which is downregulated during DC maturation [27]. According to our results, MHCIIlow cells appear to represent an immature DC phenotype with low expression of MHCII and costimulatory molecules, such as CD86.
In accordance with morphology and phenotype analysis, transcriptome analysis confirmed heterogeneity in BMDC maturation: DC-related genes were highly expressed in MHCIIhigh cells, including costimulatory molecules and innate immune receptors, whereas MHCIIlow cells showed higher levels of genes mainly expressed on HSCs. The higher expression levels of IRF4 and CCR7 in MHCIIhigh cells supported BMDCs being within the mature gate, as well as the development of subsets into cDC2s [28, 29]. In accordance with, BMDCs under the influence of GM-CSF appeared closer to cDC2s [27]. The MDS analysis revealed the differences between MHCIIhigh and MHCIIlow cells. It also showed that the few difference between the two cell populations involved the maturation state rather than being due to the presence of distinct cell types. One sample from the MHCIIlow cell population (MHCIIlow_3) was more close to the MHCIIhigh population in the MDS analysis. It is possible that variations were due to differences between individual samples, or to factors such as variable culture conditions. In further studies, it should be possible to identify their closest relatives in vivo by transcriptome analysis of in vitro-generated BMDCs from gnotobiotic miniature pigs. Furthermore, our RNA-sequencing data may provide information relevant to the investigation of porcine HSCs and BMDCs.
In addition, MHCIIhigh cells expanded into T cells and phagocytized beads more efficiently than MHCIIlow cells, with similar gene ontology enrichment of antigen presentation and innate immune receptors. Accordingly, porcine GM-CSF culture preferentially differentiated BMCs into immature (MHCIIlow cells) and mature (MHCIIhigh cells) DCs.
On the basis of morphological, phenotypical, and gene expression criteria, we classified cell two populations based on MHCII expression; we suggest that the MHCIIhigh and MHCIIlow populations can be best-classified by their maturation stage. Therefore, this study might lead to a better understanding of the function of DCs and provides useful information for future studies using porcine BMDCs.
Supporting information
S1 Fig [apc]
Adherent cells developing in bone marrow GM-CSF culture.
S1 Table [docx]
Primers used in quantitative RT-PCR.
Zdroje
1. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392(6673):245–52. Epub 1998/04/01. doi: 10.1038/32588 9521319.
2. Merad M, Sathe P, Helft J, Miller J, Mortha A. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu Rev Immunol. 2013;31:563–604. Epub 2013/03/23. doi: 10.1146/annurev-immunol-020711-074950 23516985; PubMed Central PMCID: PMC3853342.
3. Wan H, Dupasquier M. Dendritic cells in vivo and in vitro. Cell Mol Immunol. 2005;2(1):28–35. Epub 2005/10/11. 16212908.
4. Anguille S, Lion E, Tel J, de Vries IJ, Coudere K, Fromm PD, et al. Interleukin-15-induced CD56(+) myeloid dendritic cells combine potent tumor antigen presentation with direct tumoricidal potential. PLoS One. 2012;7(12):e51851. Epub 2013/01/04. doi: 10.1371/journal.pone.0051851 23284789; PubMed Central PMCID: PMC3532168.
5. Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med. 1992;176(6):1693–702. Epub 1992/12/01. doi: 10.1084/jem.176.6.1693 1460426; PubMed Central PMCID: PMC2119469.
6. Olatunde AC, Abell LP, Landuyt AE, Hiltbold Schwartz E. Development of endocytosis, degradative activity, and antigen processing capacity during GM-CSF driven differentiation of murine bone marrow. PLoS One. 2018;13(5):e0196591. Epub 2018/05/11. doi: 10.1371/journal.pone.0196591 29746488; PubMed Central PMCID: PMC5944997.
7. Rogers PB, Driessnack MG, Hiltbold Schwartz E. Analysis of the developmental stages, kinetics, and phenotypes exhibited by myeloid cells driven by GM-CSF in vitro. PLoS One. 2017;12(7):e0181985. Epub 2017/07/28. doi: 10.1371/journal.pone.0181985 28750033; PubMed Central PMCID: PMC5531556.
8. Helft J, Bottcher J, Chakravarty P, Zelenay S, Huotari J, Schraml BU, et al. GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of CD11c(+)MHCII(+) Macrophages and Dendritic Cells. Immunity. 2015;42(6):1197–211. Epub 2015/06/18. doi: 10.1016/j.immuni.2015.05.018 26084029.
9. Na YR, Jung D, Gu GJ, Seok SH. GM-CSF Grown Bone Marrow Derived Cells Are Composed of Phenotypically Different Dendritic Cells and Macrophages. Mol Cells. 2016;39(10):734–41. Epub 2016/10/30. doi: 10.14348/molcells.2016.0160 27788572; PubMed Central PMCID: PMC5104881.
10. Szulc-Dabrowska L, Struzik J, Ostrowska A, Guzera M, Toka FN, Bossowska-Nowicka M, et al. Functional paralysis of GM-CSF-derived bone marrow cells productively infected with ectromelia virus. PLoS One. 2017;12(6):e0179166. Epub 2017/06/13. doi: 10.1371/journal.pone.0179166 28604814; PubMed Central PMCID: PMC5467855.
11. Oh S, Choi EY. The Differential Role of GM-CSF and Flt3L on Myelopoiesis of Lineage Negative Bone Marrow Cells. J Immunol. 2018;200(1). PubMed PMID: WOS:000459977701077.
12. Lutz MB, Inaba K, Schuler G, Romani N. Still Alive and Kicking: In-Vitro-Generated GM-CSF Dendritic Cells! Immunity. 2016;44(1):1–2. Epub 2016/01/21. doi: 10.1016/j.immuni.2015.12.013 26789912.
13. Marquet F, Vu Manh TP, Maisonnasse P, Elhmouzi-Younes J, Urien C, Bouguyon E, et al. Pig skin includes dendritic cell subsets transcriptomically related to human CD1a and CD14 dendritic cells presenting different migrating behaviors and T cell activation capacities. J Immunol. 2014;193(12):5883–93. Epub 2014/11/12. doi: 10.4049/jimmunol.1303150 25385823.
14. Auray G, Keller I, Python S, Gerber M, Bruggmann R, Ruggli N, et al. Characterization and Transcriptomic Analysis of Porcine Blood Conventional and Plasmacytoid Dendritic Cells Reveals Striking Species-Specific Differences. J Immunol. 2016;197(12):4791–806. Epub 2016/11/12. doi: 10.4049/jimmunol.1600672 27837108.
15. Auray G, Lachance C, Wang Y, Gagnon CA, Segura M, Gottschalk M. Transcriptional Analysis of PRRSV-Infected Porcine Dendritic Cell Response to Streptococcus suis Infection Reveals Up-Regulation of Inflammatory-Related Genes Expression. PLoS One. 2016;11(5):e0156019. Epub 2016/05/24. doi: 10.1371/journal.pone.0156019 27213692; PubMed Central PMCID: PMC4877111.
16. Proll MJ, Neuhoff C, Schellander K, Uddin MJ, Cinar MU, Sahadevan S, et al. Transcriptome profile of lung dendritic cells after in vitro porcine reproductive and respiratory syndrome virus (PRRSV) infection. PLoS One. 2017;12(11):e0187735. Epub 2017/11/16. doi: 10.1371/journal.pone.0187735 29140992; PubMed Central PMCID: PMC5687707.
17. Gunzer F, Hennig-Pauka I, Waldmann KH, Mengel M. Gnotobiotic piglets as an animal model for oral infection with O157 and non-O157 serotypes of STEC. Methods Mol Med. 2003;73:307–27. Epub 2002/10/12. doi: 10.1385/1-59259-316-x:307 12375439.
18. Fairbairn L, Kapetanovic R, Sester DP, Hume DA. The mononuclear phagocyte system of the pig as a model for understanding human innate immunity and disease. J Leukocyte Biol. 2011;89(6):855–71. doi: 10.1189/jlb.1110607 WOS:000291108400007. 21233410
19. Hartmann SB, Mohanty S, Skovgaard K, Brogaard L, Flagstad FB, Emneus J, et al. Investigating the Role of Surface Materials and Three Dimensional Architecture on In Vitro Differentiation of Porcine Monocyte-Derived Dendritic Cells. PLoS One. 2016;11(6):e0158503. Epub 2016/07/01. doi: 10.1371/journal.pone.0158503 27362493; PubMed Central PMCID: PMC4928952.
20. Carrasco CP, Rigden RC, Schaffner R, Gerber H, Neuhaus V, Inumaru S, et al. Porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties. Immunology. 2001;104(2):175–84. Epub 2001/10/31. doi: 10.1046/j.0019-2805.2001.01299.x 11683958; PubMed Central PMCID: PMC1783296.
21. Summerfield A, McCullough KC. The porcine dendritic cell family. Dev Comp Immunol. 2009;33(3):299–309. Epub 2008/06/28. doi: 10.1016/j.dci.2008.05.005 18582937.
22. Hwang JH, Gupta MK, Park CK, Kim YB, Lee HT. Establishment of major histocompatibility complex homozygous gnotobiotic miniature swine colony for xenotransplantation. Anim Sci J. 2015;86(4):468–75. Epub 2014/12/11. doi: 10.1111/asj.12312 25491717.
23. Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009;25(9):1105–11. Epub 2009/03/18. doi: 10.1093/bioinformatics/btp120 19289445; PubMed Central PMCID: PMC2672628.
24. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28(5):511–5. Epub 2010/05/04. doi: 10.1038/nbt.1621 20436464; PubMed Central PMCID: PMC3146043.
25. Wilkinson-Ryan I, Kim J, Kim S, Ak F, Dodson L, Colonna M, et al. Incorporation of porcine adenovirus 4 fiber protein enhances infectivity of adenovirus vector on dendritic cells: implications for immune-mediated cancer therapy. PLoS One. 2015;10(5):e0125851. Epub 2015/05/02. doi: 10.1371/journal.pone.0125851 25933160; PubMed Central PMCID: PMC4416912.
26. Auray G, Facci MR, van Kessel J, Buchanan R, Babiuk LA, Gerdts V. Porcine neonatal blood dendritic cells, but not monocytes, are more responsive to TLRs stimulation than their adult counterparts. PLoS One. 2013;8(5):e59629. Epub 2013/05/15. doi: 10.1371/journal.pone.0059629 23667422; PubMed Central PMCID: PMC3648567.
27. Guilliams M, Ginhoux F, Jakubzick C, Naik SH, Onai N, Schraml BU, et al. Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. Nat Rev Immunol. 2014;14(8):571–8. Epub 2014/07/19. doi: 10.1038/nri3712 25033907; PubMed Central PMCID: PMC4638219.
28. Gao Y, Nish SA, Jiang R, Hou L, Licona-Limon P, Weinstein JS, et al. Control of T helper 2 responses by transcription factor IRF4-dependent dendritic cells. Immunity. 2013;39(4):722–32. Epub 2013/10/01. doi: 10.1016/j.immuni.2013.08.028 24076050; PubMed Central PMCID: PMC4110745.
29. Persson EK, Uronen-Hansson H, Semmrich M, Rivollier A, Hagerbrand K, Marsal J, et al. IRF4 transcription-factor-dependent CD103(+)CD11b(+) dendritic cells drive mucosal T helper 17 cell differentiation. Immunity. 2013;38(5):958–69. Epub 2013/05/15. doi: 10.1016/j.immuni.2013.03.009 23664832.
Článek vyšel v časopise
PLOS One
2019 Číslo 11
- Řekněte mi, co jíte, a já vám řeknu, jaká je úroveň vaší kognice
- Duševní onemocnění se u teenagerů přenáší v rámci školní třídy
- Od kostiček k lidské kůži. Jak si postavit 3D biotiskárnu?
- Pomohou nám s detekcí karcinomu plic i včely?
- Původní český test ALBA pro odhalení mírných kognitivních deficitů získal certifikaci MZ ČR
Nejčtenější v tomto čísle
- A daily diary study on maladaptive daydreaming, mind wandering, and sleep disturbances: Examining within-person and between-persons relations
- A 3’ UTR SNP rs885863, a cis-eQTL for the circadian gene VIPR2 and lincRNA 689, is associated with opioid addiction
- A substitution mutation in a conserved domain of mammalian acetate-dependent acetyl CoA synthetase 2 results in destabilized protein and impaired HIF-2 signaling
- Molecular validation of clinical Pantoea isolates identified by MALDI-TOF